Pepsin is a proteolytic enzyme produced in the mucosal lining of the stomach and acts to degrades protein, together with chymotrypsin and trypsin. During digestion, these enzymes, each of which is effective in severing links between particular types of amino acids (e.g. phenylalanine, tryptophan and tyrosine), collaborate to break down dietary proteins to their components, i.e., peptides and amino acids.
Current studies indicate that immune protection against cancer requires the generation of a potent cellular immune response against a unique tumor antigen expressed by a malignant cell. Thus, successful immune protection would first require identifying a unique antigen in the tumor cells (tumor specific antigen) and then inducing a potent T-cell response targeted to the tumor antigen. These tumor-associated antigens, however, would still be recognized by immune cells as ‘self’ molecules, and so no true activation of the immune system would occur. Thus, two obstacles in targeting these tumor-associated molecules as a vaccine include the unresponsiveness of the immune system to ‘self’ molecules, which restricts its ability to generate potent cellular immune responses, and preventing the generated immune response from being directed to normal cells that express the target antigen.
Proteins that show promise in overcoming these problems include heat shock proteins (HSPs). HSPs include a collection of ubiquitously expressed cytoprotective proteins, which are expressed by cells under conditions of cell stress, such as increased temperature, viral infection and oxidative stress. Certain HSPs have been shown to have immunomodulatory effects, such as the induction of cytokines and the promotion of cell activation and maturation (see, Pockley A G, Lancet 363 (9382) 469-476 (2003)).
For example, Zheng et al. (2001) report that cell surface targeting of HSP gp96 induces dendritic cell maturation and antitumor immunity as demonstrated by the expression of immune factors such as interleukins and certain cell surface antigens (e.g., CD40, CD80 and MHC class II antigens). It has been known for some time that heat shock proteins bind peptide and that heat shock proteins purified from cells chaperone a large number of peptides derived from the cells from which they are isolated. This is the so-called ‘antigenic repertoire’ of that cell. Studies have demonstrated that immunizing mice with HSP70, HSP90 and gp96 isolated from murine tumor cells induces anti-tumor immunity and tumor-specific cytolytic T-cells. These studies also show that the immunity results from tumor-derived peptides associated with the heat shock protein rather than from the heat shock proteins themselves. More recently, studies reported the use of calreticulin, HSP110 and grp170 in heat shock protein-based cancer immunotherapy. Specific immunogenicity of tumor-derived heat shock protein preparations have been studied in relation to fibro sarcomas, lung carcinoma, prostate cancer, spinal cell carcinoma and melanomas in mice and rats of different haplotypes. These studies included chemically-induced tumors, UV-induced tumors, and spontaneous tumors. Heat shock proteins show promise in that preparations isolated from a given cell may be associated with a range of peptides, including self and antigenic peptides and in that HSP-peptide complexes are highly immunogenic.
Certain heat shock proteins demonstrate “superantigen” activity. They are capable of activating large numbers of T-lymphocytes in a major histocompatibility complex-restricted manner. This polyclonal activation of certain T-cell subsets may be responsible for some of the immunomodulatory effects. These components have been reported to stimulate immune responses to certain neoplasms and may be involved in the pathogenesis of certain autoimmune diseases.
Gp96 is a HSP of particular interest. Gp96 is a 96 kDa glycoprotein localized to the endoplasmic reticulum, which can also be found at the cell surface. Gp96 is released into the extra cellular space during necrotic cell death and activates dendrite cells and macrophages by realizing inflammatory cytokines and inducing dendrites cells to mature. Gp96 has the ability to transfer antigenic peptides for their MHC-class I-restricted presentation and allows gp96 to function as an efficient messaging system alerting the immune system of an infection. This includes the receptor-mediated uptake of gp96 by dendrite cells. The receptor is CD91, which is known as the α2 macroglobulin (α2M) receptor expressed on phagocytes. The presentation of gp96-associated peptide by antigen-presenting cells (“APC's”) is induced by α2 macroglobulin. Gp96 is bound by CD91 on dendrite cells and internalized. Gp96 induces the expression of co-stimulatory molecules and the release of interleukin 12 (IL-12) and tumor necrotic factor α (“TNFα”) by the APC.
Certain infections, such as by the human immunodeficiency virus, have also presented challenges in targeting the disease-causing organism and neutralizing it. Typically, infection with the human immunodeficiency virus, HIV-1, eventually causes acquired immunodeficiency syndrome (AIDS) and an associated syndrome, AIDS-related complex (ARC). Neutralizing this virus has proved difficult, largely because its structure obstructs immune system access to viral epitopes and its genetic material is highly variable. Accordingly, researchers have been seeking prophylactic and therapeutic methods for preventing or controlling HIV which are not dependent upon antibody-mediated immunity.
The HIV retrovirus replicates in certain immune system cells, specifically the CD4+ subset of T-lymphocytes (pre-Th cells arising in the thymus). In the usual course of a cell-mediated immune response to an intracellular pathogen such as a virus, dendritic cells (antigen-presenting cells) carrying antigen fragments and secreted cytokines activate these CD4+ T-cells. Activated cells, called T-helper or Th cells, in turn secrete their own cytokines and stimulate macrophages. CD4+Th cells also propagate cellular immune response by binding chemotactic cytokines (chemokines, CCs) to their CC surface receptors. It is by this route that HIV-1 infection of these cells is enabled because, in addition to binding chemokines, these CC receptors act together with the CD4+ surface glycoprotein as co-receptors for HIV-1 and mediate entry of the virus into the CD4+Th cell. There, the virus usurps the native genetic material for viral replication while destroying cell functions essential for building immunity; the increasing destruction of these cells appears to be responsible for the eventual collapse of the cell-mediated immune system often seen in terminal AIDS patients.
It has been recognized that denying entry into CD4+ cells to the HIV-1 virus could at least slow the progress of the infection and alleviate, if not cure, the disease and/or its symptoms. The complex mechanism by which the virus crosses the cell membrane has been widely investigated. Broadly, the entry of human immunodeficiency virus into, for example, CD4+ Th1 cells (T-helper type 1 cells), is dependent upon a sequential interaction of the gp120/gp41 subunits of the viral envelope glycoprotein gp160 with the CD4+Th1 cell surface glycoprotein and the cell surface receptor CCR5. On binding of gp120 with its cell surface binding sites, a conformational change in the latent gp41 subunit through an intermediate state to an active state is initiated, inducing fusion of the viral and cellular membranes and transport of the virus into the cell (Weissenhom et al., Nature, 387:426-30 (1997)).
Accordingly, numerous binding experiments have been conducted in an effort to find antiviral ligands that will effectively compete with the HIV-1 for CD4+ gp and/or CCR5 binding sites, or that will preferentially block gp120 and/or gp41 binding domains. In one example, a reported structure (X-ray crystallography) comprising an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding, is said to reveal a CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change on CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for viral immune evasion, “which should guide efforts to intervene” (Nature 393 (6686):632-1, 1998). Also, it has been shown that inhibition of the change in structure of gp41 from its intermediate to active state with peptides used as competitors for critical cell receptors may reduce viral load, and that while gp120 masks epitopes on the gp41 subunit in its latent state, gp41 may be vulnerable to neutralizing antibodies in its transient or intermediate state (Molecular Membrane Biology 16:3-9, 1999).